Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Techniques for improving activity based biosensors: a Kuhl platform for engineering(Montana State University - Bozeman, College of Agriculture, 2020) Thomas, Merrilee Anne; Chairperson, Graduate Committee: Thomas Hughes and Susy C. Kohout (co-chair); Thomas E. Hughes was a co-author of the article, 'Optically activated, customizable, excitable cells Kuhl platform for evolving next gen biosensors' submitted to the journal 'PLOS One' which is contained within this dissertation.According to Kuhn, ''there are three classes of problems - determination of significant facts, matching of facts with theory, and articulation of that theory (Kuhn 2012).'' The current paradigm in molecular neuroscience is that there is a need for revolutionary tool development in neuroscience. Interestingly, the need for better tools in neuroscience is to answer neuroscience theories and provide the determination and articulation of those theories. Currently, the neuroscientist's toolbox is growing and the ways in which those tools are used is rapidly changing. Neuroscience underwent a revolution when we were able to take single-cell recording in vivo and then assign field properties to individual neurons based upon those responses (O'Keefe and Bouma 1969; O'Keefe and Dostrovsky 1971; Moser et al. 1995). Scientists became adept at imaging increasingly smaller regions of the functioning human brain (Price 2012). We have since been able to genetically encode and manipulate proteins and pathways while recording from them using fluorescence (Southern and Berg 1982; Chalfie 2009). In vitro and in vivo we have harnessed the use of light to stimulate or inhibit specific neurons or ligands (Boyden 2011; Adamantidis et al. 2007). These tools are just the beginning and by no means is this an exhaustive list. We introduce the Kuhl synthetic cell system that provides a customizable de-novo excitable cell. The Kuhl system is activated using a blue light photo activated cyclase bPAC. It can be used to create better tools to image the brain and can be used to screen multi-color fluorescent sensors. Interestingly, sensors that are within bPACs activation spectrum can be used in these synthetic cells. We show that both red and green Ca 2+ sensors can be imaged simultaneously, and both Ca 2+ and Voltage sensors can be screened in the Kuhl system. The Kuhl system has the potential to be used to screen for drug compounds and in theory, they could be used in studying pathways that are less understood, such as the mTOR pathway.Item Improving osseointegration of PEEK through surface textures(Montana State University - Bozeman, College of Engineering, 2019) Scott, Renn Patrick; Chairperson, Graduate Committee: Cecily RyanPEEK (Polyetheretherketone) is one of the most promising alternatives to titanium in cortical bone implants due to being biologically inert and having an elastic modulus similar to that of bone. It also has favorable reactions conducive to common medical imaging methods such as X-ray and magnetic resonance imaging (MRI) as compared to commonly used metals such as titanium and stainless steel. However PEEK is not inherently osseoconductive, leading to longer healing times and a greater chance of infection. Many different methods exist for improving osteoblast growth, such as the addition of bio-active materials like hydroxyapatite. Manipulating the surface texture of PEEK could provide better environments for cells to attach and can be used as another layer with other techniques, making the tissue interface more robust. The main objective of this project is to observe cell adhesion to a textured surface to identify cell preference for surface geometry as a first step to improve full integration of non-resorbable implants into bone tissue. The methods explored were also chosen for their repeatability, reliability and lack of chemical modification compared to other successful surface modulation techniques. The surface textures were embossed into PEEK using micro-etched aluminum molds. Textures vary in their shape, spacing, size, depth and surface convexity/concavity. The cell adhesion was recorded through fluorescent confocal microscopy and the cell-substrate interaction was observed under electron microscopy. The results were that 25 micron and 10 micron features discouraged cell adhesion while 325 micron and 120 micron features encouraged cell adhesion with pillars performing better than holes. The best feature was the 325x40 micron square pillars. With a cell volume to surface area ratio of 5.13, a live cell count of 276.5, a dead cell count of 9.00, and a non-dimensional distance to feature of 0.67.Item The interstitial fluid pressure response during stress-relaxation of articular cartilage due to viscosity and porous media effects: a computational study(Montana State University - Bozeman, College of Engineering, 2018) Paschke, Brandon James; Chairperson, Graduate Committee: Erick JohnsonArticular cartilage is a complex material made of several fluid and solid components. A model that fully describes the responses of cartilage is required to accurately create a cartilage replacement that can be used in cases of injury or disease. Modeling of articular cartilage has proven difficult and currently no constitutive law fully describes its solid and fluid responses. Many of the current models describe the interstitial fluid as inviscid, even though it is known that proteoglycan migration within cartilage causes a viscous response within interstitial fluid. The goal of this research was to create a viscous fluid porous media model that better captures the compressive resistance of cartilage created by migration of interstitial fluid during cartilage compression. Through the creation of this model it was possible to capture the experimental magnitudes of fluid pressure within cartilage during unconfined slow compression simulations. As part of this model, a porous media approximation was used, which demonstrates that small variations in the solid matrix, comprised of collagen fibers, can cause large variations in system response. Magnitudes of mean pressure values, after 150 seconds of compression, for the viscous fluid porous media model bound the values found in experimental testing. Limitations of the fluid model are that system relaxation isn't captured and the slope increase of pressures during compression for experiments don't match those of the fluid model. A main conclusion drawn from the model is that viscosity of interstitial fluid plays a large role in creating compressive resistance within articular cartilage. Another takeaway is that the porous media approximation greatly impacts the magnitude of fluid pressurization, which creates a need to accurately represent the solid matrix within cartilage.Item Biological redesign of virus particles for a new era of catalytic materials(Montana State University - Bozeman, College of Letters & Science, 2016) Jordan, Paul Campion; Chairperson, Graduate Committee: Trevor Douglas; Dustin P. Patterson, Kendall N. Saboda, Ethan J. Edwards, Heini M. Miettinen, Gautam Basu, Megan C. Thieleges and Trevor Douglas were co-authors of the article, 'Self-assembling biomolecular catalysts for hydrogen production' in the journal 'Nature chemistry' which is contained within this dissertation.; Joseph C-Y Wang, Ethan J. Edwards, Heini M. Miettinen, Amanda L. Le Sueur, Megan C. Thielges , Adam Zlotnick and Trevor Douglas were co-authors of the article, 'Redesign of a virus particle for NADH-driven hydrogen production' which is contained within this dissertation.; This dissertation contains one article of which Paul Campion Jordan is not the main author.Biology has designed a suite of compartments and barriers that confine fundamental biochemical reactions. Such barriers include the membrane-bound organelles but also a suite of protein-based compartments that architecturally and chemically integrate catalytic processes. These compartments co-polymerize from multiple protein subunits to form polyhedral structures that spatially separate enzymatic processes. Protein compartments confine volatile intermediates, trap toxic reaction products, and co-localize multiple enzymatic processes for catalytic enhancements. The protein-based compartments represent, advantageously, a combination of form and function that has inspired the synthesis of new, designer materials. The self-assembly of cage-like structures, the structures of which are reminiscent of the compartments, has been used for the directed encapsulation of active enzymes. We have used the capsid from bacteriophage P22, as a nanocontainer for directing the encapsulation of a variety of gene products, including active enzymes. The P22 capsid assembles from a coat protein (CP) and a scaffold protein (SP) which templates its assembly. Using the simplicity of the P22 expression system, a strategy was developed and implemented for the directed encapsulation of an active, [NiFe] hydrogenase. We hypothesized and proved the enzyme active site needed to be matured by accessory proteins found within the expression host. A two plasmid expression system was designed, where the hydrogenase cargo was under the control of a different inducer than the P22 CP. The [NiFe]-hydrogenase is a heterodimer and each enzyme subunit was fused to different SP. The resultant packaging of the two SP fusions, with the hydrogenase large and small subunits fused to them stabilized a weak heterodimeric structure. Remarkably, the stabilizing effects of the capsid allowed us to probe the infrared signatures associated with the hydrogenase active site. Finally, the progress made here in developing a virus capsid for H2 production left room to build increased complexity into the P22-Hydrogenase system while also taking inspiration from the innate, biological function of the hydrogenase. We incorporated a cytochrome/cytochrome reductase pair to drive H 2 production using NADH. These designs, built at the molecular level, represent inherently renewable catalysts that pave the way for a new era of catalytic materials synthesized entirely by biology.